u251 glioblastoma cancer cell line  (ATCC)

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: (A–B) Western blot analysis of uPAR and cathepsin B expression in U251 and 5310 cells 72 hrs after transfection with SV, pU, pC and pCU. (C) Quantitative analysis of uPAR and cathepsin B expression by densitometry. Results from three independent experiments are shown as mean ± SD (** p <0.001). GAPDH was used as a loading control. (D) Cell cycle distribution of U251 and 5310 cells. Propidium iodide-stained cells were analyzed for DNA content using flow cytometry. (E) Histograms represent the percentage of cells in G0/G1, S and G2/M phases. The data represent one of three independent experiments. Values are mean ± SD of three different experiments (** p <0.001). (F) Cells were stained for apoptosis using TdT-mediated dUTP nick end-labeling (TUNEL) assay. Data shown are from a representative experiment. (G) Quantification of apoptotic cells expressed as percent of DAPI-stained cells. Bars represent the mean ± SD of three experiments ( *p <0.05; **p <0.001). (H) Analysis of mitochondrial membrane potential using MitoLight kit. Cells were collected, incubated with MitoLight dye for 20 min at room temperature, and observed for fluorescence; red fluorescence indicates healthy cells while green fluorescence indicates apoptotic cells.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Western Blot, Expressing, Transfection, Staining, Flow Cytometry, End Labeling, TUNEL Assay, Incubation, Fluorescence

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: (A) Expression of pro- and anti-apoptotic molecules in U251 and 5310 cells 72 hrs after transfection with SV or pCU. Human apoptosis antibody arrays were exposed to cell lysates and processed as per the manufacturer's instructions. (B) Densitometric analysis and graphical representation of fold change of pro- and anti-apoptotic molecules. (C) Western blot analysis of Bcl-2 and Bax expression in U251 and 5310 cells 72 hrs transfection. The blots were stripped and re-probed with GAPDH antibody to verify equal loading. The experiments were repeated three times and representative blots are shown. (D) Densitometric analysis showing the Bcl-2/Bax ratio in U251 and 5310 cells. Columns: mean of triplicate experiments; bars: SE; ** p <0.001. (E) Semi-quantitative RT-PCR analysis of Bcl-2 and Bax mRNA expression in pU-, pC- and pCU-transfected U251 and 5310 cells. Total RNA was extracted 72 hrs after transfection, and cDNA was synthesized as described in . PCR was set up using first-strand cDNA as the template for Bcl-2, Bax and GAPDH. (F) Densitometric analysis showing the Bcl-2/Bax mRNA ratio. Columns: mean of triplicate experiments; bars: SE; ** p <0.001, significant difference from untreated control or SV-transfected control.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Synthesized

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: (A) FACS analysis of pCU-transfected U251 and 5310 cells pretreated with 40 µM broad caspase inhibitor (Z-Asp-2, 6-dichlorobenzoylmethylketone). (B) Histograms represent the percentage of cells in G0/G1, S and G2/M phases. The data represent one of the three independent experiments. Values are mean ± SD of three different experiments (* p <0.05, ** p <0.001). uPAR and cathepsin B downregulation induced activation of caspase-9, caspase-3, ICAD, CAD in pU-, pC- and pCU-treated U251 cells. (C) Western blot analysis for active caspase-9, caspase-3, ICAD and CAD in U251 cells. (D) Expression of cytochrome c in mitochondrial and cytosolic fractions was determined by western blot analysis. Cytochrome oxidase IV was used as a marker for mitochondrial fractions (MF); GAPDH was used for cytosolic fractions (CF). (E) Immunoprecipitation of Apaf-1 from U251 cell lysates. Total cell lysates were subjected to immunoprecipitation using anti-caspase-9 antibody and then immunoblotted for Apaf-1. Total lysates from SV-, pU-, pC- and pCU-treated U251 cells were separated into mitochondrial and nuclear fractions as per standard protocols and immunoblotted for AIF. (F) Expression levels of AIF in mitochondrial (MF) and nuclear fractions (NF) were determined by western blot analysis. Cytochrome oxidase IV was used as a marker for mitochondrial fractions; laminin B was used for nuclear fractions. Total lysates from SV-, pU-, pC- and pCU-treated 5310 cells were fractionated into mitochondrial and cytosolic fractions as per standard protocols and immunoblotted for cytochrome c. Immunoblots are representative of three experiments.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Transfection, Activation Assay, Western Blot, Expressing, Marker, Immunoprecipitation

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: U251 and 5310 cells were transfected with SV, pU, pC or pCU. Untreated cells served as the control. After 72 hrs, cells were collected, and total cell lysates were prepared and western blotted as per standard protocol using normal and phosphorylated forms of PI3K, Akt and PDGFR-β. (A–B) Expression of normal and phosphorylated forms of PI3K, Akt and PDGFR-β in U251 and 5310 cells. Columns: mean of triplicate experiments; bars: SE; * p <005 and ** p <0.001, significant difference from untreated control or SV-transfected control. Effect of PI3K inhibitor (Wortmannin) and PDGFR tyrosine kinase inhibitor (PTKI) on expression of p-PDGFR-β, p-PI3K p85α, Bcl-2 and Bax. U251 and 5310 cells were treated with 20 and 40 µg/mL of PI3K inhibitor (Wortmannin;WN) for 48 hrs, and the expression levels of p-Akt, p-CREB, Bcl-2 and Bax were determined by western blotting using appropriate antibodies. (C–D) Western blot analysis of p-AKT, p-CREB, Bcl-2 and Bax expression levels after treatment with the PI3K inhibitor. Separately, U251 and 5310 cells were treated with 5 nM and 10 nM PDGFR tyrosine kinase inhibitor (PTKI) for 48 hrs. Expression levels of p-PDGFR-β, p-PI3K, p-Akt, p-CREB, Bcl-2 and Bax were determined by western blotting using appropriate antibodies. (E–F) Western blot analysis of p-PDGFR-β, p-PI3K, p-Akt, p-CREB, Bcl-2 and Bax expression levels in U251 and 5310 cells treated with PDGFR tyrosine kinase inhibitor.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Transfection, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: U251 and 5301 cells were transfected with SV, pU, pC or pCU for 72 hrs. Cell lysates were separated into nuclear (NF) and cytoplasmic fractions (CF) and immunoblotted for p-CREB. Total cell lysates were probed for CREB. (A–B) Western blot analysis of p-CREB in nuclear and cytoplasmic fractions of U251 and 5310 cells. Nuclear extracts were prepared from SV- (control) and pCU-transfected U251 and 5310 cells and supershift analysis of CREB binding activity was carried out using EMSA. For supershift analysis, nuclear extracts were incubated with supershift specific CREB antibody (2.0 µg) or IgG (2.0 µg) prior to incubation with CREB binding buffer. (C–D) Supershift analysis of CREB DNA binding activity in U251 and 5310 cells. The experiments were performed three times and representative blots are shown. Nuclear extracts were prepared from SV-, pU-, pC- and pCU-transfected U251 and 5310 cells, and DNA binding activity of CREB was determined by colorimetric assay using the TransAM ELISA kit. To test specificity, DNA binding activity was tested in the presence of an excess of oligonucleotide containing a wild-type or mutated CREB consensus binding site. (E–F) Inhibition of DNA binding activity of CREB by nuclear extracts of pU-, pC- and pCU-treated U251 and 5310 cells. The bars represent the mean ± SD of three different experiments. *Statistically different compared to controls and pU-, pU- and pCU-treated groups (** p <0.001).

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Transfection, Western Blot, Binding Assay, Activity Assay, Incubation, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: PLoS ONE

Article Title: Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

doi: 10.1371/journal.pone.0013731

Figure Lengend Snippet: Intracranial tumors were established in nude mice by injecting U251 and 5310 cells that were treated with SV and pCU as described in . (A–B) The brains were embedded in paraffin, sectioned and stained for apoptosis by TdT-mediated nick end-labeling (TUNEL) followed by DAB staining. Nuclei were counterstained with methyl green. Data shown are representative of five fields. Brown stain around green nuclei indicates apoptotic cells. (C–D) Immunohistochemical analysis of uPAR, cathepsin B, Bcl-2 and Bax was performed in paraffin-embedded U251 and 5310 tumor sections. Appropriate protein-specific antibodies were used. Fields with brown staining as a result of DAB interaction were scored for protein expression. (E–F) uPAR and cathepsin B, Bcl-2 and Bax expression was detected in tumor tissue lysates from intracranial tumors of mice that received SV and pCU. Results are representative of three separate experiments.

Article Snippet: We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study.

Techniques: Staining, End Labeling, TUNEL Assay, Immunohistochemical staining, Expressing

Journal: Chemical Science

Article Title: Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors

doi: 10.1039/d3sc01315d

Figure Lengend Snippet: Cytotoxicity assay of alkoxyamines in U87-MG and U251-MG cells in 2D culture. (A) Survival of U251-MG and U87-MG cells measured by MTT-assay in 2D-cultures, 72 h after treatment with alkoxyamines. (B) IC 50 values of alkoxyamines measured by MTT-assay in U87-MG and U251-MG cells. Values are the average of at least three independent experiments ± sem. (C) Correlation between IC 50 values and half-life time of homolysis at 37 °C of the alkoxyamines.

Article Snippet: The U87-MG and U251-MG human glioblastoma cancer cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Cytotoxicity Assay, MTT Assay

Journal: Chemical Science

Article Title: Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors

doi: 10.1039/d3sc01315d

Figure Lengend Snippet: Impact of ALK4, ALK5, ALK9 and ALK13 on the migration potential of U87-MG and U251-MG cells after 12 hours of treatment. Representative pictures of migrated (A) U87-MG and (B) U251-MG cells without treatment and after 12 hours of treatment with ALK4, ALK5, ALK9 and ALK13. Scale bar = 150 μm. Quantification of the anti-migratory effect of ALK4, ALK5, ALK9 and ALK13 in (C) U87-MG and (D) U251-MG cells. Results are expressed as a relative percentage of migrated cells after 12 hours of treatment versus migrated cells non-exposed to treatment. Values are the average of at least three independent experiments ± sem. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The U87-MG and U251-MG human glioblastoma cancer cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Migration

Journal: Chemical Science

Article Title: Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors

doi: 10.1039/d3sc01315d

Figure Lengend Snippet: Evaluation of the efficacy of ALK4, ALK5, ALK9 and ALK13 at 10 μm in U251-MG and U87-MG GBM spheroids. Monitoring of the growth of U251-MG spheroids (A and B) and U87-MG spheroids (D and E) by fluorescence quantification for 4 days after treatment with ALK4, ALK5, ALK9 and ALK13 at 5 μm and 10 μm. treatment efficacy was expressed as a percentage of spheroid growth inhibition vs. control spheroids. Values are the average of at least three independent experiments ± sem. * p < 0.05; ** p < 0.01; *** p < 0.001. Representative pictures over time of (C) U251-MG and (F) U87-MG spheroids non-treated or exposed to ALK4 at 10 μm. Images were obtained by fluorescence microscopy ( λ ex 580 nm/ λ em 620 nm). Scale bar = 250 μm.

Article Snippet: The U87-MG and U251-MG human glioblastoma cancer cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Fluorescence, Inhibition, Microscopy

Journal: Chemical Science

Article Title: Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors

doi: 10.1039/d3sc01315d

Figure Lengend Snippet: Study of the intracellular incorporation of ALK4 in GBM cells. Structure of (A) ALK4-FITC and (B) ALK17-FITC. (C) Fluorescence calibration range of ALK4-FITC and ALK17-FITC measured by spectrofluorimetry (PHERAstar, λ ex 495 nm/ λ em 519 nm). (D) Pictures of U251-MG cells observed by fluorescence microscopy (40X objective) after 2 hours of treatment with ALK4-FITC and ALK17-FITC at 15 μm. Left panel: nuclear staining by DAPI ( λ ex 340 nm/ λ em 450 nm) and ALK4-FITC analysis within cells ( λ ex 495 nm/ λ em 519 nm). Right panel: nuclear staining by DAPI ( λ ex 340 nm/ λ em 450 nm) and mitochondrial network analysis by using mtDsRed fluorescence ( λ ex 580 nm/ λ em 620 nm). Scale bar = 50 μm.

Article Snippet: The U87-MG and U251-MG human glioblastoma cancer cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Fluorescence, Microscopy, Staining

Journal: Chemical Science

Article Title: Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors

doi: 10.1039/d3sc01315d

Figure Lengend Snippet: Study of the mechanism of action of ALK4. (A) Cell survival measured by MTT-assay after 72 hours of treatment with ALK4, its nitroxide SG1 and its alkyl radical on inactive forms PEA and PEA-OH, in 2D-culture of U251-MG cells. (B) Measurement of glioblastoma and medulloblastoma spheroid growth 4 days after treatment with SG1 at 100 μm compared to control spheroid growth. (C) Survival of U251-MG cells co-exposed to ALK4 and the alkyl scavenger troxerutin (72 hours) in 2D-culture. (D) Superoxide production in 2D-cultures of U251-MG cells upon treatment with ALK4 alone and in combination with troxerutin at 25 μm for 6 hours. (E) Mitochondrial network of U87-MG, U251-MG, ONS-76 and UW228-2 cells observed by fluorescence microscopy (40X objective, mtDsRed fluorescence, λ ex 580 nm/ λ em 620 nm) 6 hours after treatment with 5 μm and 10 μm of ALK4. (F) Measurement of apoptosis induction in U251-MG cells after a 48 hours-treatment with ALK4. Scale bar = 50 μm. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The U87-MG and U251-MG human glioblastoma cancer cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: MTT Assay, Fluorescence, Microscopy

Journal: Chemical Science

Article Title: Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors

doi: 10.1039/d3sc01315d

Figure Lengend Snippet: Evaluation of the efficacy of the bioconjugates ALK4-CHYMOp and ALK4-MMPp in GBM spheroids. (A) Structure of the bioconjugates ALK4-CHYMOp and ALK4-MMPp. (B) Monitoring of the growth of U251-MG spheroids by fluorescence quantification for 4 days after treatment with ALK4-CHYMOp and with ALK-MMPp. Treatment efficacy was expressed as a percentage of spheroid growth inhibition vs. control spheroids. Values are the average of at least three independent experiments ± sem. * p < 0.05; ** p < 0.01; *** p < 0.001. (C) Representative pictures over time of U251-MG spheroids non-treated or exposed to 50 μm of ALK4-CHYMOp and ALK4-MMPp. Images were obtained by fluorescence microscopy ( λ ex 580 nm/ λ em 620 nm). Scale bar = 250 μm. (D) Measurement of GBM and MB spheroid growth 4 days after treatment with MMPp peptide (100 μm) compared to control spheroid growth.

Article Snippet: The U87-MG and U251-MG human glioblastoma cancer cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Fluorescence, Inhibition, Microscopy

Journal: Chemical Science

Article Title: Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors

doi: 10.1039/d3sc01315d

Figure Lengend Snippet: Evaluation of the bioconjugate ALK4-MMPp in GBM and MB organotypic models. Representative pictures over 14 days of (A) U251-MG and (C) ONS-76 organotypic models non-treated or exposed to 100 μm of ALK4-MMPp (treatment at D0, D4, D7 and D10). Images were obtained using the JuLi™Stage live imaging system ( λ ex 580 nm/ λ em 620 nm, stitching mode 5 × 6). Scale bar = 2500 μm. Monitoring of the growth of (B) U251-MG and (D) ONS-76 spheroids in organotypic brain and cerebellar models by fluorescence quantification for 14 days after treatment with ALK4-MMPp. Values are the average of at least three independent experiments ± sem. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The U87-MG and U251-MG human glioblastoma cancer cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Imaging, Fluorescence

Journal: Chemical Science

Article Title: Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors

doi: 10.1039/d3sc01315d

Figure Lengend Snippet: Analysis of the toxicity of ALK4 and ALK4-MMPp. (A) HES staining and yH2AX immunostaining of U251-MG organotypic model after 14 days of treatment with the bioconjugate ALK4-MMPp. Scale bar = 100 μm. (B) Study of toxicity of ALK4 and ALK4-MMPp in zebrafish, 5 fishes per well. Values are the average of at least three independent experiments ± sem. (C) Representative pictures of live (left) and dead (right) zebrafishes.

Article Snippet: The U87-MG and U251-MG human glioblastoma cancer cell lines were obtained from ATCC (Manassas, VA, USA).

Techniques: Staining, Immunostaining